Peptoid affinity ligands

a technology of affinity ligands and protein ligands, which is applied in the field of affinity ligands of proteins, can solve the problems of low chemical and biochemical stability, high cost of antibody isolation and purification, and high cost of protein ligands, and achieves high affinity and selectivity for antibodies, wide chemical diversity, and high target specificity and affinity. the effect of high affinity and selectivity

Active Publication Date: 2016-03-17
LIGATRAP TECH LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0003]Extensive research has been carried out in both industry and academia to discover inexpensive and robust ligands with high affinity and selectivity for antibodies. A variety of synthetic compounds, including triazinic scaffolds, amino acids and peptides have been proposed for the selection of affinity ligands for downstream processing of biotherapeutics. A class that has recently been considered for the design of affinity ligands is represented by peptoids. This class of compounds possesses ideal characteristics for such application. First, the display of functional groups on peptoids resembles that of peptides, implying that peptoids can be designed or selected with levels of affinity and selectivity comparable to those of peptide ligands. Further, owing to the so-called “sub-monomer” protocol of synthesis, which employs primary amines, peptoids can explore a much wider chemical diversity than that available to protein ligands and peptides comprising natural amino acids. This enables fine tuning their composition to achieve higher target specificity and affinity. Finally, peptoids are completely resistant to proteolysis and are therefore advantageous for the purification of antibodies from fluids containing active enzymes, like whole plasma and its fractions or lysates of cell cultures, plants and other organisms.

Problems solved by technology

However, therapies based on antibodies are very expensive to consumers.
Their high price is due in part to the high cost of isolation and purification of these biomolecules.
Despite their high selectivity for IgG, these protein ligands suffer from high cost and low chemical and biochemical stability.
Protein ligands show in general poor chemical resistance towards the alkaline (0.1-1.0 M NaOH) cleaning-in-place and sanitisation-in-place procedures periodically applied for the removal of contaminants and required by regulatory guidelines.
Further, they are prone to proteolytic degradation by enzymes present in the feed.
Both chemical and enzymatic agents can cause ligand degradation and leakage of ligand fragments from the resin, resulting in shorter column lifetime and potential presence of toxic and immunogenic leachates in the product mainstream.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Solid-Phase Synthesis of a Tetramer Peptoid Ligand and Use of the Resulting Affinity Adsorbent for the Purification of Human Polyclonal or Monoclonal Antibodies

[0076]The selected groups, as numbered from the N- to the C- terminus, are: imidazoyl, indolyl-, guanidyl-, and methyl-. Correspondingly, the amines employed for the peptoid synthesis are respectively histamine, tryptamine, agmatine, and methylamine. Presented in Scheme 1, as follows:

The chromatographic resin Toyopearl AF-Amino-650M is chosen as solid support for synthesis. The synthesis comprises the following steps:[0077]1. Coupling of bromoacetic acid in N,N′-dimethylformamide (DMF) via diisopropylcarbodiimide activation (hereafter referred to as DIC). Wash the resin with DMF and equilibrate with NMP.[0078]2. Coupling of methylamine in N-methylpyrrolidone (NMP). Wash the resin with NMP and equilibrate with DMF.[0079]3. Coupling of bromoacetic acid by DIC. Wash the resin with DMF and equilibrate with NMP.[0080]4. Coupling o...

example 2

Preparation of Peptoids Directly to Amine-Containing Resin

[0087]Preparation Procedures: Peptoids were synthesized directly onto Toyopearl AF-amino 650M resin (Tosoh Biosciences) at a 0.1 mmol / mL loading with a Biotage Alstra automated peptide synthesizer under microwave assistance using methods described previously (Fara et al. Tet. Lett. 2006 47, 1011-1014; Olivos et al. Org. Lett. 2002, 4(23), 4057-4059). The Fmoc-protected monomers for glycine, N-(-3-guanidinpropyl)glycine, N-(isobutyl)glycine, N-(3-(Boc-amino)-propyl)glycine and N-(benzyl)glycine were coupled to the peptoid using N,N,N′,N′-Tetramethyl—O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU) with diisopropylethyl amine as the coupling agent in dimethyl formamide for 30 minutes at 50 C (Huang et al. PNAS 2012, 109(49), 19922-19927.). After coupling, the secondary amine was deprotected using 20% piperidine in DMF. The remaining residues were coupled to the peptoid sequence using a submonomer approach. Chloroaceti...

example 3

Binding of Human Immunoglobulin from PBS in Absence of Other Proteins

[0094]The ability of the peptoid affinity ligands to bind human IgG molecules was tested in batch mode. A 50% slurry of the resin was prepared, of which 100 μl (˜50 mg resin) was transferred into a spin column. The resin was washed with 2×200 μL of 0.1 M glycine buffer (pH 2.5), and rinsed with phosphate buffer saline (PBS), pH 7.4 prior to chromatography. Human polyclonal immunoglobulin G (hIgG) was diluted to 0.5 mg / mL with PBS. After draining the resin, the hIgG solution was added to the resin to obtain a ratio of 2 mg hIgG per mL of resin, and mixed vigorously for 5 minutes at room temperature. After incubation, the resin was centrifuged at 500×g for 2 min and the filtrate was collected and labeled as “flow-through” fraction. The resin was washed with 2×200 μL of PBS. Elution buffer (0.1M glycine, pH 2.5, 200 μL) was added to the resin and mixed vigorously for 5 minutes at room temperature. After centrifugation...

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Abstract

Compounds of Formulas I:and shorter variants thereof are described, along with solid supports having such compounds coupled thereto, and the use thereof as affinity ligands for antibodies.

Description

RELATED APPLICATION DATA[0001]This application claims the benefit of and priority from U.S. Provisional Patent Application No. 62 / 084,383, filed on Nov. 25, 2014, and is a continuation-in-part of International Application No. PCT / US2014 / 039995, filed on May 29, 2014, which claims the benefit of and priority from U.S. Provisional Patent Application No. 61 / 829,712, filed on May 31, 2013, the disclosures of each of which are incorporated herein by reference in their entirety.BACKGROUND OF THE INVENTION[0002]Monoclonal antibodies and Fc-fusion proteins have emerged as an important class of therapeutic proteins for the treatment of a number of unmet diseases such as cancer, autoimmune diseases, immunodeficiency, skin disorders and neurological disorders. These products account for 40% of the overall pharmaceutical market with a volume of $35 billion in 2011. However, therapies based on antibodies are very expensive to consumers. Their high price is due in part to the high cost of isolati...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K1/22B01J20/24B01J20/26B01J20/286C07K16/00
CPCC07K1/22C07K16/00B01J20/286B01J20/24B01J20/261
Inventor MENEGATTI, STEFANO
Owner LIGATRAP TECH LLC
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